Method for preparing human neoplastically transformed cells

ABSTRACT

A method for preparing neoplastically transformed cells from human-derived cells, including the step of introducing human telomerase catalytic subunit (hTERT) gene, SV40 small T antigen (SV40ST) gene, and an antisense oligonucleotide derived from human 28S rRNA into the human-derived cells. The method for preparing neoplastically transformed cells from human-derived cells can be utilized when a variety of human normal cells are induced to be neoplastically transformed in order to elucidate cancer onset mechanisms, so that the method can be effectively utilized in search of target molecules for a new medicament.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for preparing humanneoplastically transformed cells. More specifically, the presentinvention relates to a method for preparing neoplastically transformedcells from human-derived cells using antisense RNA derived from human28S rRNA, cells obtained by the method, a kit for use in the method, anda method of using the cells obtained by the method in assessment ofpharmacologically efficacy of an anticancer agent.

2. Discussion of the Related Art

From the studies of human-derived cells using an oncogene encoding a DNAtumor virus protein, such as a Simian virus 40 (SV40) large T antigen,an adenovirus E1A or E1B, Human papillomavirus E6 or E7, a model ofcarcinogenesis presumes that malignant transformation of human culturedcells consists of two phases of an immortalization process and atumorigenetic process.

In the immortalization process, telomeric sequences are added to endparts of chromosomes by telomerase activity, thereby making it possibleto acquire an infinite lifespan. Therefore, if immortalization takesplace, a clonal expansion of cells takes place, so that opportunity foradditional genetic alterations is increased, thereby increasing apossibility of tumorigenetic conversion. In addition, in thetumorigenetic process, the immortally transformed cells acquire anautonomous growth potential in the absence of certain growth factors.

Generally, cancer cells express uncertain phenotypes different fromnormal cells, e.g. genomic instability, lack of responsiveness to thechemical signalings, and loss of differentiation. However, it yetremains obscure whether those alterations involved in the tumorigeneticprocess described above result from cumulative mutations in genesrelated to the particular proteins, or a cascade of gene expressioninitiated by a factor activated in the onset of tumorigenesis aboutwhich little is known.

In recent years, it has been reported that normal human cells can betransformed by expression of human telomerase catalytic subunit (hTERT),SV40 early region (ER), and activated H-ras gene (See W. C. Hahn et al,“Creation of human tumour cells with defined genetic elements.” letter,Nature, 29 Jul. 1999). According to the report, in the presence ofhTERT, the SV40 large T antigen (LT) inactivates retinoblastoma protein(pRB) tumor suppressor pathway and p53 tumor suppressor pathway, and theSV40 small T antigen (ST) serves to dysfunction phosphatase 2A. Also,the activated ras gene is known to participate in the tumorigenecity oftransformed cells. Therefore, the aberrations are suggested to be theminimal number of genetic events required in the malignant conversion ofhuman-derived cells.

In addition, Japanese Patent Laid-Open No. 2008-109889 discloses amethod for inducing neoplastic transformation of human-derived cells,including expressing Src in the presence of hTERT and an SV40 large Tantigen (LT).

On the other hand, the present inventors have previously reported that anucleotide sequence resulting from addition of a poly(A) chain to acertain nucleotide sequence at a 3′-end side of a first stem of asecondary structure of U5 small nuclear RNA [also named as transformingRNA (TR)] is expressed as non-coding mRNA, thereby making it possible todrive rat fibroblastic 3Y1 cells into the neoplastically transformedcells, i.e. tumor cells (See K. Hamada, “Morphological transformationcaused by a partial sequence of U5 small nuclear RNA.” Mol. Carcinog.,1997, 20, 175-188). The 3Y1 cells had already acquired the ability toproliferate infinitely through spontaneous immortalization. Therefore,it is deduced that the TR plays a role in tumorigenesis, not inimmortalization; as a result of further studies in view of the above,the present inventors have found that the above-mentioned transformationis greatly dependent on a certain polypurine sequence ggagaggaa (SEQ IDNO: 3) of the TR.

Furthermore, it is reported that as a result of studies using a rabbitreticulocyte extract, the TR sequence synthesized in vitro directlybinds to 28S rRNA of ribosome in the nascent chain elongation of peptidesynthesis to affect secretory signal peptide-associated translation (K.Hamada et al, “Effect of Transforming RNA on the Synthesis of a Proteinwith a Secretory Signal Sequence in Vitro.” J. Biol. Chem., 1999,274(22), 15786-15796). Moreover, it is found that theoligodeoxynucleotides (ODNs) containing the above-mentioned polypurinesequence bind to cucc sequence (SEQ ID NO: 4) on 28S rRNA, and on theother hand that the oligodeoxynucleotides (ODNs) containing an antisensesequence of the above polypurine sequence bind to gagg (nucleotides 48to 51) (SEQ ID NO: 5) on 7SL RNA of signal recognition particle (SRP).In view of the above, it is considered that the chain elongation arrestactivity influences physiological actions between the 28S rRNA and theSRP RNA.

However, it has been found that the above-mentioned TR cannot be inducedinto neoplastically transformed cells by transforming human-derivedcells in the presence of hTERT, depending upon a combination with c-myc,an activated H-ras, or a gene encoding SV40 Large T or the like.

An object of the present invention is to provide a method for preparingneoplastically transformed cells from human-derived cells, cellsobtained by the method, a kit for use in the method, and a method ofusing cells obtained by the method in assessment of pharmacologicalefficacy of an anticancer agent, which are free from any safetyproblems.

These and other objects of the present invention will be apparent fromthe following description.

SUMMARY OF THE INVENTION

As a result of intensive studies in order to solve the above problems,the present inventors have found that a certain antisense short chainRNA complementary to human 28S rRNA binds on a ribosome surface as ahuman-specific transforming RNA (hTR), so that human-derived cells canbe transformed and induced into neoplastically transformed cells, andthe present invention is perfected thereby.

In sum, the present invention relates to:

[1] a method for preparing neoplastically transformed cells fromhuman-derived cells, including the step of introducing human telomerasecatalytic subunit (hTERT) gene, SV40 small T antigen (SV40ST) gene, andan antisense oligonucleotide derived from human 28S rRNA intohuman-derived cells;[2] a method for introducing a gene for neoplastically transforminghuman-derived cells, including incorporating human telomerase catalyticsubunit (hTERT) gene, SV40 small T antigen (SV40ST) gene, and anantisense oligonucleotide derived from human 28S rRNA into the same ordifferent vectors, and introducing the genes into human-derived cellstherewith;[3] neoplastically transformed cells derived from human-derived cells,obtained by the method as defined in the above [1];[4] a kit for use in the method as defined in the above [1], containinghuman telomerase catalytic subunit (hTERT) gene, SV40 small T antigen(SV40ST) gene, and an antisense oligonucleotide derived from human 28SrRNA;[5] a method for screening an anticancer agent, including the steps ofculturing neoplastically transformed cells from human-derived cellsobtained by the method as defined in the above [1] in the presence orabsence of a candidate compound, and judging that the candidate compoundhas a high possibility of having an action as an anticancer agent in acase where the neoplastically transformed cells that are cultured in thepresence of the candidate compound have a larger degree of inhibition ofneoplastic transformation from human-derived cells, as compared to theneoplastically transformed cells that are cultured in the absencethereof; and[6] a method for screening an anticancer agent, including the steps ofculturing human-derived cells into which the genes are introducedaccording to the method as defined in the above [2] in the presence orabsence of a candidate compound, and judging that the candidate compoundhas a high possibility of having an action as an anticancer agent in acase where the neoplastically transformed cells that are cultured in thepresence of the candidate compound have a larger degree of inhibition ofneoplastic transformation from human-derived cells, as compared to theneoplastically transformed cells that are cultured in the absencethereof.

According to the method for preparing neoplastically transformed cellsfrom human-derived cells of the present invention, human-derived cellscan be more safely induced into neoplastically transformed cells havinga potent neoplastically transforming ability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is views showing the results of interaction between human 28SrRNA and antisense short RNAs. FIG. 1-a shows a putative secondarystructure of domain 5 of human 28S rRNA, wherein the left side is a V8-4region, and the right side is the entire domain 5, and wherein the lowerrows show nucleotide sequences of asR70 (SEQ ID NO: 1), asR56 (SEQ IDNO: 6), asR46 (SEQ ID NO: 2), and sR70 (SEQ ID NO: 7). FIG. 1-b is aview showing the assessment results of cleavage with RNase H whenco-cultured with the oligonucleotide (ON), wherein a left side shows theresults of rabbit lysates, and a right side shows the results of humancell extracts. FIG. 1-c shows binding properties of asR70, asR56, andsR70, wherein as to asR70, starting from left, the results of the asR70probe alone, untreated RNA (2.0×10⁵ cpm of the asR70 probe being added),ODN-treated RNA (2.0×10⁵ cpm of the asR70 probe being added), untreatedRNA (1.0×10⁵ cpm of the asR70 probe being added), ODN-treated RNA(1.0×10⁵ cpm of the asR70 probe being added) are shown; as to asR56,starting from left, the results of the asR56 probe alone, ODN-treatedRNA (2.0×10⁵ cpm of the asR56 probe being added), ODN-treated RNA(1.0×10⁵ cpm of the asR56 probe being added), untreated RNA (2.0×10⁵ cpmof the asR56 probe being added) are shown; and as to sR70, starting fromleft, the results of the sR70 probe alone, ODN-treated RNA (2.0×10⁵ cpmof the sR70 probe being added), ODN-treated RNA (1.0×10⁵ cpm of the sR70probe being added), and untreated RNA (2.0×10⁵ cpm of the sR70 probebeing added) are shown. Here, the results of one in which total RNA istreated with 20 units of RNaseH.

FIG. 2-a is views showing the morphologies of the transformed cells andcolony formation in a soft agar medium, wherein the left side showscells transformed with hTERT, SV40ST, and asR70 (R7S), and the rightside shows cells transformed with hTERT and asR70 (R7), and wherein anupper row is views showing cell morphologies, and a lower row is viewsshowing colony formation. FIG. 2-b is views showing, starting from anupper row, the results of hTERT (TRAP assay), the results for asR70(RT-PCR), and the results of SV40ST (immunoblotting). FIG. 2-c is graphsshowing influences on the growth of the transformed cells of theoligonucleotides derived from ribosome. The graphs show the cellcondition after 4.5 months from the introduction, wherein the left graphshows cell growth, and the right graph shows cell density. FIG. 2-dshows the results of colony formation test of the transformed cells. Thephotographs show general examples of each of large colonies, middlecolonies, and small colonies, starting from left.

FIG. 3 is views showing the results of spectral karyotype (SKY) imageanalysis of transformed cells.

FIG. 4 is views showing the results of inhibition of the Bip proteinsynthesis and the induction of endoplasmic reticulum stress response.FIG. 4-a is views showing the results of assessing synthesizing rates ofBip, fibronectin, and integrin β1, according to pulse-chase labeling andimmunoprecipitation using ³⁵S-methionine. FIGS. 4-b and 4-c are viewsshowing the results of immunoblotting of Bip, eIF2α, phosphorylatedeIF2α, and ATF4.

DETAILED DESCRIPTION OF THE INVENTION

The method for preparing neoplastically transformed cells fromhuman-derived cells includes the step of introducing human telomerasecatalytic subunit (hTERT) gene, and SV40 small T antigen (SV40ST) geneinto human-derived cells, and the method has a great feature in that theantisense oligonucleotide derived from human 28S rRNA is used togetherduring the step.

The V8-4 region of the domain 5 is located at an about 3 kb-region fromthe 5′ end of human 28S rRNA (see FIG. 1-a). The V8 region correspondsto yeast ES27 that has been reported to have an action of movingnonribosomal factors such as chaperones, modifying enzymes, SRP ortranslocon (protein transport device on the endoplasmic reticulummembrane) from the L1 side on the ribosome toward the tunnel exit site.In addition, the V8-4 region contains six cucc sequences (SEQ ID NO: 4)in the latter half of the 5′ end side. Therefore, as a result ofcomprehensively judging these phenomena, the present inventors haveconsidered that short RNA complementary to ES27 of human 28S rRNA can beused as human transforming RNA (hTR), and that when the hTR is usedtogether with hTERT and SV40ST, they have surprisingly found out thathuman-derived cells can be transformed. The resulting transformed cellsacquired anchorage-independent growth potential and showed chromosomalaberrations, thereby making it possible to introduce hTERT and SV40STwith the hTR, which suggests that the human-derived cells can betransformed into neoplastically transformed cells.

The method for preparing neoplastically transformed cells fromhuman-derived cells of the present invention includes the step ofintroducing human telomerase catalytic subunit (hTERT) gene, SV40 smallT antigen (SV40ST) gene, and an antisense oligonucleotide derived fromhuman 28S rRNA into human-derived cells.

The term “gene” as used herein refers to a factor that plays a role ofgene information of an organism, which is meant to be used to includeDNA and RNA. In addition, the term “nucleotide” is meant to be used toinclude DNA and RNA. Also, the term “antisense oligonucleotide” refersto an oligonucleotide having a nucleotide sequence complementary to acertain nucleotide sequence (hereinafter referred to as sense sequence),and capable of hybridizing to the sense sequence.

The human telomerase catalytic subunit (hTERT) gene is not particularlylimited, and includes those known in the field of art. The telomerase isan enzyme that maintains a telomeric length by being antagonistic to theshortening of the telomeric length by cell division. The telomerasecontains as constituents RNA serving as a template of the telomericsequence, and a reverse transcriptase, and this reverse transcriptasemoiety is hTERT.

The SV40 small T antigen (SV40ST) gene is not particularly limited, andincludes those known in the field of art. The SV40 (Simian virus 40) isa virus belonging to Polyomavirus, and is separated from Rhesus monkeykidney cells, and this virus produces a large T antigen and a small Tantigen as early stage proteins at an early infection stage to cells(before DNA synthesis takes place). In the present invention, the smallT antigen is used as an antigenic virus.

The antisense oligonucleotide derived from human 28S rRNA includes anantisense oligonucleotide of a sense sequence in the expansion segment27 (ES27) region of human 28S rRNA. Among them, an oligonucleotidecontaining the nucleotide sequence shown in SEQ ID NO: 1, anoligonucleotide containing the nucleotide sequence shown in SEQ ID NO:2, and an oligonucleotide containing the nucleotide sequence shown inSEQ ID NO: 6 are preferred; an oligonucleotide essentially consisting ofthe nucleotide sequence shown in SEQ ID NO: 1, an oligonucleotideessentially consisting of the nucleotide sequence shown in SEQ ID NO: 2,and an oligonucleotide essentially consisting of the nucleotide sequenceshown in SEQ ID NO: 6 are more preferred; and an oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID NO: 1 and anoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 2 are even more preferred.

In addition, in the present invention, as the above-mentionedoligonucleotide, an oligonucleotide having deletion, addition, insertionor substitution of one or more nucleotides in the above-mentionednucleotide sequence is also preferably used. Concretely, theoligonucleotide includes an oligonucleotide having homology of at least70% or more, preferably 80% or more, more preferably 90% or more, andeven more preferably 95% or more, to the oligonucleotide shown in theabove-mentioned nucleotide sequence, or an oligonucleotide containingthe oligonucleotide. Also, the nucleotide length is not particularlylimited, and the nucleotide length is preferably from 40 to 200, morepreferably from 40 to 150, even more preferably from 40 to 100, evenmore preferably from 40 to 90, even more preferably from 40 to 80, andeven more preferably from 46 to 70. The oligonucleotide as describedabove exhibits similar effects (capable of neoplastically transforminghuman-derived cells at an equivalent level) to the cases of using theoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 1, the oligonucleotide consisting of the nucleotide sequence shownin SEQ ID NO: 2, and the oligonucleotide consisting of the nucleotidesequence shown in SEQ ID NO: 6. Here, the term homology as used hereincan be obtained by, for example, using a search program BLAST in whichan algorithm developed by Altschul et al. (The Journal of MolecularBiology, 215, 403-410 (1990)) is employed.

A schematic view of an oligonucleotide consisting of the nucleotidesequence shown in SEQ ID NO: 1, an oligonucleotide consisting of thenucleotide sequence shown in SEQ ID NO: 2, and an oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID NO: 6 is shown inFIG. 1. These oligonucleotides are an antisense oligonucleotide to anoligonucleotide shown by 3001 to 3070 nucleotides (hereinafter alsoreferred to as asR70), an antisense oligonucleotide to anoligonucleotide shown by 3015 to 3060 nucleotides (hereinafter alsoreferred to as asR46), and an antisense oligonucleotide to anoligonucleotide shown by 3015 to 3070 nucleotides (hereinafter alsoreferred to as asR56), each located in the domain 5 of human 28S rRNA.All the oligonucleotides are complementary to a sense sequence locatedin the expansion segment 27 (ES27, 2889 to 3551 nucleotides) region,which are suggested to partake in the function of ES27, in other words,proliferation. Here, the abbreviation asR as used herein means antisenseshort RNA, and the abbreviation sR means sense short RNA.

The method for synthesizing an antisense oligonucleotide derived fromhuman 28S rRNA is not particularly limited, and a phosphoramiditemethod, a phosphorothioate method, a phosphotriester method or the like,using a known oligonucleotide synthesizer can be employed.

The gene and the antisense oligonucleotide mentioned above may be amodified or substituted product in accordance with a known method withinthe range so as not to markedly lower its activity, in order to increasestability and affinity to the cells. For example, the gene and theantisense oligonucleotide can be also used in the form of a derivativeformed by substituting a phosphate group, or a hydroxyl group orhydroxyl groups of a ribose moiety, with another stable group.

The human-derived cells into which the gene and the antisenseoligonucleotide described above are introduced are not particularlylimited, and include human fibroblast cells, human endothelial cells,human epithelial cells, and the like. The derivations or tissues of theabove-mentioned cells are not particularly limited.

The method of introducing the gene and the antisense oligonucleotidementioned above into human-derived cells is not particularly limited.For example, a product obtained by incorporating the gene and theantisense oligonucleotide mentioned above into any vector can be used.

It is preferable that the vector is self-replicable in a host cell, andat the same time contains, in addition to the gene and the antisenseoligonucleotide mentioned above, a promoter and a transcriptiontermination sequence. In addition, the vector may contain a genecontrolling a promoter. Here, the promoter is not particularly limitedso long as the gene and the antisense oligonucleotide mentioned abovecan be expressed in a host cell.

The preferred vector usable in the present invention includes, forexample, adenoviral vector, Vaccinia virus vector, retrovirus vector andthe like.

The gene and the antisense oligonucleotide mentioned above may beintroduced at the same time, collectively or individually, and they mayeach be introduced at a different timing. The gene and the antisenseoligonucleotide mentioned above may be introduced into separate vectorsand used, or may be introduced into the identical vector and used. In acase where the gene and the antisense oligonucleotide incorporated intoseparate vectors are used, for example, those in which hTERT gene isincorporated into pBABE vector, those in which SV40ST gene isincorporated into pLHCX vector, and those in which an antisenseoligonucleotide is incorporated into pLPCX vector can be used. Also, ina case where the gene and the antisense oligonucleotide incorporatedinto the identical vector are used, a preferred vector includesretroviral vector, and the locations of the gene and the antisenseoligonucleotide mentioned above in the vector are not particularlylimited, and for example, the gene and the antisense oligonucleotide arelocated between a promoter and a transcription termination sequence, inthe order of hTERT gene, SV40ST gene, and the antisense oligonucleotide,starting from the promoter side.

The method for introducing a vector includes an electroporation method,a calcium phosphate method, a lipofection method and the like.

In addition, the vector may be introduced by preparing a vector obtainedby further incorporating viral DNA for infection to the introduced cellsinto the above-mentioned vector, thereby infecting human-derived cellswith the vector-introduced virus. The virus for infection includesadenovirus, adeno-associated virus, retrovirus, and the like.

Here, the method of constructing the vector, a concrete method of usingthe vector, and the like may be referred to, for example, textbooks suchas Sambrook, J., et. al., Molecular Cloning: A Laboratory Manual; 2^(nd)Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor. N.Y., 1989.

Thus, hTERT gene, SV40ST gene, and an antisense oligonucleotide derivedfrom human 28S rRNA can be introduced into human-derived cells. Thecells obtained are formed into transformed cells which express a proteinencoded by hTERT and SV40ST, and at the same time express the antisenseoligonucleotide derived from human 28S rRNA. Therefore, the presentinvention provides cells obtained by introduction of the hTERT gene, theSV40ST gene, and the antisense oligonucleotide derived from human 28SrRNA. The cells may be cultured under conditions appropriate for thecells to repeat population doubling, and screened with a known agent.Here, in the present invention, those obtained by introducing SV40STgene and an antisense oligonucleotide derived from human 28S rRNA intohTERT immortalized cells into which hTERT gene is already incorporatedare also embraced within the scope of the present invention. Thecondition of human-derived cells in which neoplastic transformation isinduced by hTERT gene, SV40ST gene, and an antisense oligonucleotidederived from human 28S rRNA can be confirmed in accordance with a methoddescribed in Examples set forth below.

In addition, in the present invention, human-derived cells can beneoplastically transformed by using hTERT gene, SV40ST gene, and anantisense oligonucleotide derived from human 28S rRNA in combination, sothat a kit containing hTERT gene, SV40ST gene, and an antisenseoligonucleotide derived from human 28S rRNA can be used in theneoplastic transformation of human-derived cells. The present inventionalso provides a kit containing hTERT gene, SV40ST gene, and an antisenseoligonucleotide derived from human 28S rRNA.

Furthermore, the neoplastic transformation of human-derived cells isfacilitated by using hTERT gene, SV40ST gene, and an antisenseoligonucleotide derived from human 28S rRNA in combination, and since adegree of neoplastic transformation is at a certain level, human-derivedcells that are neoplastically transformed using hTERT gene, SV40ST gene,and an antisense oligonucleotide derived from human 28S rRNA, orhuman-derived cells into which hTERT gene, SV40ST gene, and an antisenseoligonucleotide derived from human 28S rRNA are introduced, can be usedin screening an anticancer agent. Therefore, the present inventionprovides a method of screening an anticancer agent. The above-mentionedmethod is an in vitro assessment in the human system, assumption to invivo assessment is facilitated, so that an effective substance can befound more conveniently and quickly.

A concrete method includes a method including the steps of:

contacting a candidate compound with the neoplastically transformedcells obtained by introducing hTERT gene, SV40ST gene, and an antisenseoligonucleotide derived from human 28S rRNA into human cells, and

judging that the candidate compound has a high possibility of having anaction as an anticancer agent in a case where the cells after contactingthe candidate compound has a smaller degree of neoplastic transformationthan the cells before contacting the candidate compound.

More specifically, there are two embodiments:

an embodiment of culturing neoplastically transformed cells fromhuman-derived cells obtained by introducing hTERT gene, SV40ST gene, andan antisense oligonucleotide derived from human 28S rRNA intohuman-derived cells in the presence or absence of a candidate compound,and judging that the candidate compound has a high possibility of havingan action as an anticancer agent in a case where the neoplasticallytransformed cells that are cultured in the presence of the candidatecompound have a larger degree of inhibition of neoplastic transformationfrom human-derived cells, as compared to the neoplastically transformedcells that are cultured in the absence thereof (Embodiment 1); and

an embodiment of comprising culturing human-derived cells into which theabove genes are introduced in the presence or absence of a candidatecompound, and judging that the candidate compound has a high possibilityof having an action as an anticancer agent in a case where theneoplastically transformed cells that are cultured in the presence ofthe candidate compound have a larger degree of inhibition of neoplastictransformation from human-derived cells, as compared to theneoplastically transformed cells that are cultured in the absencethereof (Embodiment 2).

In Embodiment 1 and Embodiment 2, the conditions for culturing the cellsin the presence or absence of the candidate compound can be carried outin the same manner according to known conditions when culturing thecells before the above genes are introduced, except for the presence orabsence of the candidate compound, and are not particularly limited.

In Embodiment 1, when it is judged that the candidate compound has ahigh possibility of having an action as an anticancer agent, in a casewhere a part of the cells cultured in the presence of the candidatecompound is found to undergo changes such as transformed morphologicallosses, induction of apoptosis, and anchorage-independent losses, ascompared to the cells cultured in the absence thereof, a candidatecompound to which the cells are contacted has an action as an anticanceragent, so that the candidate compound can be judged to have an effect ofreducing neoplastic transformation.

In Embodiment 2, when it is judged that the candidate compound has ahigh possibility of having an action as an anticancer agent, in a casewhere a part of the cells cultured in the presence of the candidatecompound is found to have a smaller degree of population doubling andchange in cell morphologies, as compared to the cells cultured in theabsence thereof, a candidate compound to which the cells are contactedhas an action as an anticancer agent, so that the candidate compound canbe judged to have an effect of reducing neoplastic transformation.

EXAMPLES

The present invention will be explained hereinbelow on the basis ofExamples, without intending to limit the present invention to theseExamples and the like. Here, hTERT gene was obtained from Dr. F.Ishikawa or Addgene 1774, SV40ST gene was obtained from JCRB gene bank(pMTI0D), and the oligonucleotide derived from human 28S rRNA wassynthesized herein or obtained from Takara Bio Inc., and each was used.In addition, human fibroblast cells (MJ90 cells) were obtained from Dr.O. M. Pereira-Smith, and human fibroblast cells (TIG3 cells) wereobtained from RB cell bank, and used.

Reference Example 1 Binding Properties of ggagaggaa Sequence (SEQ ID NO:3)

To 10 μL of a rabbit lysate or 20 μL of a human-derived cell extract wasadded a polypurine sequence ggagaggaa (SEQ ID NO: 3), a partialnucleotide sequence of transforming RNA (TR), and allowed to react inthe presence of RNase H. Thereafter, total RNA was collected andelectrophoresed on polyacrylamide gel. The results are shown in FIG.1-b. As a result, it can be seen that the ggagaggaa sequence (SEQ ID NO:3) is bound to 28S rRNA.

Reference Example 2 Binding Properties of asR70, asR56, and sR70

The binding properties of asR70, asR56, and sR70 were assessed inaccordance with the following method. The results are shown in FIG. 1-c.Concretely, first, plasmids pG-asR70, pG-asR56, and pG-sR70 wereprepared by insertions thereof into a plasmid pGEM3 (manufactured byPromega) cleaved with HindIII-XbaI, and labeled RNA probes (riboprobes)of asR70, asR56, and sR70 were produced from the plasmids obtained usingSP6 RNA polymerase (manufactured by Takara Bio Inc.) and [α-³²P]-GTP(manufactured by Perkin-Elmer). The probes obtained were electrophoresedon polyacrylamide gel, and as a result, the asR70 probe had a length of89 nucleotides (first lane from left), the asR56 probe had a length of75 nucleotides (eighth lane from right), and the sR70 probe had a lengthof 89 nucleotides (fourth lane from right).

Next, 20 μL of the human cell lysate and 4 μg of an oligodeoxynucleotideggagaggaa (SEQ ID NO: 3) were mixed, and cleaved with RNase H, andthereafter the collected total RNA was mixed with riboprobes of asR70,asR56, and sR70, and treated with RNase A and RNase T₁. Here, as acomparison, an example in which the oligodeoxynucleotide ggagaggaa (SEQID NO: 3) was not used was also treated in the same manner.

The oligodeoxynucleotide-treated total RNA showed two cleavage sites inthe binding with the asR70 probe. In other words, it was found that theoligodeoxynucleotide ggagaggaa (SEQ ID NO: 3) has binding sites within3001 to 3070 nucleotides of 28S rRNA (third to fifth lanes from left).Concretely, it could be seen that the cucc sequence (SEQ ID NO: 4, site1), corresponding to 3063 to 3066 nucleotides of 28S rRNA and the cucucusequence (SEQ ID NO: 8, site 2), corresponding to 3035 to 3040nucleotides thereof could be bound. In addition, asR56 shorter by 14nucleotides was found to be bound in the same sites (sixth to seventhlanes from right). The sites 1 and 2 are located approximately 23 to 24nucleotides apart from each other, in which the site 1 is located at ahairpin loop of the V8-4 region, and the site 2 is located at a bulgedpart, whereby suggesting that asR70 and asR56 are bound to 28S rRNA atthe above two sites. On the other hand, the binding of sR70 probe and28S rRNA could not be confirmed (second and third lanes from right). Inaddition, it is shown in K. Hamada et al, J. Biol. Chem., 1999, 274(22),15786-15796 that the gagagagag sequence (SEQ ID NO: 9), a modifiedsequence of the oligodeoxynucleotide as shown in SEQ ID NO: 3, isincapable of inhibiting the secretory protein synthesis, whereby it issuggested therefrom that the protein synthesis in the V8-4 region ismore regulated in the site 1 than in the site 2 by the interaction withRNA of SRP. Here, asR56 contains a sequence in asR70 that binds to thesite 2, and it is deduced that the binding is weak with aberrantbinding.

Example 1 Morphologies and Neoplastic Transformation of TransformedCells

Cell morphologies were observed with a phase contrast microscope forMJ90 cells transformed with hTERT, asR70, and SV40ST(hTERT+asR70+SV40ST, also simply R7S), and MJ90 cells transformed withhTERT and asR70 (hTERT+asR70, also simply R7). In addition, a colonyformation test was also conducted in a soft agar medium. The results areshown in FIG. 2-a.

Here, the transformed cells prepared in the following manner were used.Concretely, pGEM3 (manufactured by Promega) inserted with asR70 wascleaved with HindIII-EcoRI, and a fragment obtained was inserted intopLPCX (manufactured by Clontech) to prepare a vector named pLPCX-asR70.Also, an SV40ST fragment amplified by PCR was inserted into pLHCX(manufactured by Clontech) to prepare a vector named pLHCX-ST. As theplasmids for hTERT expression, pcDNAhTERTn2 (provided by Dr. F.Ishikawa) and pBABE-neo-hTERT (Addgene plasmid 1774) were used. Next,293T cells (obtained from Riken Cell Bank) were transfected withpLPCX-asR70 and pLHCX-ST, together with a retrovirus packaging plasmidpCL-10A1 (manufactured by Imgenex), the virus was then collected, andthereafter the MJ90 cells were infected with the virus. In a case wherepcDNAhTERTn2 was used, the MJ90 cells were transfected withlipofectamine 2000. In a case where pBABE-neo-hTERT was used, the viruswas prepared therewith, and TIG3 cells were infected with the virus. Thecells obtained were subjected to drug screening to select the cellshaving a life span longer than the parental cells to be used in theexperiment.

It can be seen from FIG. 2-a that cells R7S transformed with hTERT,SV40ST, and asR70 had indistinct boundaries between the cells, and thatcolonies were formed even in a soft agar medium at days 14 to 18 aftertransplantation, so that the transformed cells had anchorage-independentgrowing ability and underwent neoplastic transformation.

Example 2 Expression of hTERT, SV40ST, and asR70 in Transformed Cells

Intact normal human fibroblastic cells (MJ90), hTERT-immortalized cells(T8), cells co-transfected with hTERT and asR70 (R7), and clones thereof(R7-1 and R7-2) were furnished. In addition, cells prepared byintroduction of SV40ST into the cells R7 (R7S), clones thereof (R7S-1,R7S-2, and R7S-3), and subclones thereof (R7S-1s, R7S-2s, and R7S-3s)were also furnished. Here, as for all the furnished cells, cell linesselected in the same manner as in Example 1 were used. In addition, allthe cell lines were subjected to a colony formation test in the samemanner as in Example 1. As a result, the cells of R7S series wereconfirmed to form many colonies, but no colonies were formed at all inthe cells of R7, R7-1, and R7-2, the cells MJ90, and the cells T8.

The telomerase activity (hTERT activity) was assessed by carrying outTRAP (telomeric repeat amplification protocol) assay using PCR, with a 1μg extract of each of the cells. The results are shown in FIG. 2-b (anuppermost row of the PCR results). The cells other than the cells MJ90were shown to have telomerase activity. Here, “IC” in the figure is aninternal standard material for PCR.

The asR70 was assessed by carrying out RT-PCR using the followingasR70-specific primers (second row from top of the PCR results in FIG.2-b). As a result, 117 bp amplified products were found in the cellsother than the cells MJ90 and the cells T8.

pLPCX-retroF: (SEQ ID NO: 10) 5′-cgctagcgctaccggactc-3′ asR70-R:(SEQ ID NO: 11) 5′-ataaagcttctcgctccctccccaccc-3′

In addition, in order to confirm that the 117 bp amplified products wasspecific to asR70 cDNA, amplification was carried out by PCR using theabove-mentioned pLPCX-retroF primer, and

pLPCX-seqR: 5′-tggggtctttcattccc-3′ (SEQ ID NO: 12)as a primer specific to the non-transcriptional region of the plasmidharboring asR70 DNA; however, 233 bp amplified products could not beconfirmed, so that it could be confirmed that the asR70 is not derivedfrom the plasmid but incorporated therein (third row from top of the PCRresults of FIG. 2-b). Further, in order to show that the amplifiedamount is in an equal amount, an amplified amount of β-actin of 356 bpwas assessed (lowermost of the PCR results of FIG. 2-b). Here, “M” inFIG. 2-b stands for a molecular weight marker (HaeIII digest marker),and “*” stands for the results of amplification of plasmid pLPCX-asR70.

forward: (SEQ ID NO: 13) 5′-tgaagtgtgacgtggacatccgc-3′ reverse:(SEQ ID NO: 14) 5′-gccaatctcatcttgttttctgcgc-3′

Expression of asR70 was confirmed in the cells other than the cells MJ90and the cells T8 from the above, and it was clarified that expressionwas not derived from plasmids, but derived from those incorporated bytransformation.

Also, SV40ST was assessed by immunoblotting of the cytoplasm. Theresults are shown in the lower rows of FIG. 2-b (immunoblottingresults). As a result, expression of SV40ST of 15 to 20 kD was confirmedin the cells R7S, R7S-1, R7S-2, R7S-3, R7S-1s, R7S-2s, and R7S-3s. Here,in all the cells, as the control, expression of actin was also confirmed(bottom of the immunoblotting results in FIG. 2-b).

Example 3 Influences on the Growth of Transformed Cells ofOligonucleotides Derived from Human 28S rRNA

The hTERT-immortalized cells T8 used in Example 2 were cultured for 13months, and thereafter stored in liquid nitrogen. Thereafter, the cellswere thawed, replicated several times, and used in the followingexperiment.

Into the above-mentioned cells T8 was introduced intact pLPCX (pLPCX),asR70-incorporated pLPCX (asR70), asR56-incorporated pLPCX (asR56),asR46-incorporated pLPCX (asR46), or sR70-incorporated pLPCX (sR70),respectively, and the cells obtained were subjected to drug screeningwith puromycin to select the introduced cells T8, pLPCX, asR70, asR56,asR46, and sR70. The cell growth and the cell density (n=3) wereassessed for cell lines after 4.5 months from the introduction. Theresults are shown in FIG. 2-c. Here, the cell growth can be assessed bymeasuring an increase in cell counts with time of the cells adjusted toa certain number, and the cell density can be assessed by measuring thecell counts occupying a certain area.

From FIG. 2-c, in the cells after 4.5 months from the introduction,asR70-introduced cells and asR46-introduced cells had large cell growingrates, and high cell densities. It is suggested from the above thatasR70 and asR46 influence the cell growth mechanisms.

Example 4 Colony Formation of Transformed Cells

Into the hTERT-immortalized cells T8 used in Example 2 was introducedintact pLPCX (pLPCX), asR70-incorporated pLPCX (asR70),asR56-incorporated pLPCX (asR56), asR46-incorporated pLPCX (asR46), orsR70-incorporated pLPCX (sR70), respectively, and thereafter the cellsother than the cells introduced with intact pLPCX were further subjectedto introduction of SV40ST-incorporated pLPCX. Subsequently, the cellswere subjected to drug screening with puromycin, to select theintroduced cells T8, pLPCX, asR70, asR56, asR46, and sR70. The cellsobtained were cultured, and subjected to a colony formation test withthe passage of time (n=3). Also, in a case where colony formation wasfound, the colonies were classified according to their sizes, as “large(l), i.e. a size of 10 times or more of the cells, “middle (m), i.e. asize of 5 to 9 times or so of the cells,” and “small (s), i.e. a size of3 to 4 times or so of the cells.” A total number of colonies of largecolonies and middle colonies, i.e. l+m, and the number of smallcolonies, i.e. s, were counted, and an average thereof was obtained. Theresults are shown in FIG. 2-d. In FIG. 2-d, “NP” indicates a case wherecolonies were found but no proliferation or growth was confirmed, and“NC” indicates a case where the number of colonies was not counted.

It can be seen from FIG. 2-d that the colony formation in the soft agarwas confirmed in the cells asR70 and the cells asR46, so that both ofthe cells underwent neoplastic transformation. Also, it is suggestedthat the cells asR46 have a higher neoplastic transformation efficiencythan the cells asR70 because the former cells formed a larger number ofcolonies than the latter. It is suggested from these results that asR70and asR46 exhibit even higher transformation efficiencies. In addition,it could be confirmed that human diploid fetal lung-derived fibroblastTIG3 cells can be transformed by using asR46, hTERT, and SV40ST (theresults not shown). The above cells asR46, hereinafter also referred toas R4S, were isolated, and the clones thereof (R4S-1, R4S-2, and R4S-3)were prepared, and used in the subsequent experiment.

Example 5 Spectral Karyotyping Analysis of Transformed Cells

Five cells each of hTERT-immortalized cells (T8), cells cotransfectedwith hTERT and asR70 (R7), cells obtained by introducing SV40ST into thecells T8 (T8+ST), and clones of the cells obtained by introducing SV40STinto the cells R7 (R7S-1, R7S-2, and R7S-3) used in Example 2, and theclones of the cells obtained by introducing asR46 and SV40ST into thehTERT-immortalized cells T8 (R4S-1, R4S-2, and R4S-3) used in Example 4were subjected to spectral karyotyping analysis (SKY, reverse DAPIbanding). The results are shown in Table 1, and representative SKYstaining images are shown in FIG. 3.

TABLE 1 Karyotype [No. of abnormal karyotypes/ Cells 5 cells analyzed]T8 46, XY [5] R7 46, XY [5] T8 + ST 46, XY [5] R7S-1 46, XY, −11, + der(11)t(11; 12)(q21; ?) [5] R7S-2 45, XY, del(5)(p?), der (6) t(6; 22)(p?;q?), i (12)(q10), 22 [5] R7S-3 46, XY, −11, + der (11)t(11; 12)(q21; ?)[5] R4S-1 type A, 46, XY [3] B, 92, XXYY, del(1)(p32) [1] C, 92, XXYY,del(2)(p11) [1] R4S-2 type A, 46, XY [1] B, 46, XY, t(1; 17)(q12; p12)[1] C, 46, XY, del(13)(q22), del(17)t(17; 20)(p12; p12) [1] D, 46, XY,der(17)t(17; 20)(p12; p12) [1] E, 90, XXYY, der(8)t(1; 8)(p12; q25),t(17; 19)(p11; q13) [1] R4S-3 type A, 46, XY, der(17)t(7; 17)(q22;p12)t(17; 22)(q25; q13), der(22)t(17; 22)(q25; q13) [2] B, 91, XXYY,der(2)del(2)(q10)t(2; 18)(q10; ?), der(17)t(7; 17)(q22; p12)t(17;22)(q25; q13) × 2, −18, der(22)t(17; 22)(q25; q13) × 2 [1]

From Table 1 and FIG. 3, the cells T8, the cells R7, and the cells T8+STare confirmed to have 46 chromosome numbers, so that it can be seen thatthe cells are normal diploid cells. On the other hand, while the cellsR7S-1 and the cells R7S-3 are found to have 46 chromosome numbers,apparently, genome rearrangements took place at one site. In addition,the cells R7S-2 were confirmed to have 45 chromosome numbers, one lessthan the normal cells, and genome rearrangements were confirmed at 4sites. Also, all of the cells R4S-1, R4S-2, and R4S-3 had diversifiedchromosome numbers and sites at which genome rearrangements took place,even while the cells were derived from the same cell line, so thatseveral types thereof were confirmed. Among them, the cells R4S-1,R4S-2, and R4S-3 have larger alterations than the cells R7S-1, R7S-2,and R7S-3; however, since normal cells also exist in the cells R4S-1 andR4S-2, it is suggested that these alterations took place after thetransformation. It is suggest from the above that cells transfected withhTERT, SV40ST and asR70 are different from the normal cells or cellscotransfected with hTERT and asR70 in the genetic information in thecells.

Example 6 Induction of Endoplasmic Reticulum Stress Response inTransformed Cells

Expression of substances showing endoplasmic reticulum stress responsewas confirmed on cells T8, pLPCX, asR70, asR56, asR46, and sR70,obtained in the same manner as in Example 4. Also, as to the cellsasR70, the same experiments for those cells obtained in the same manneras in Example 2, i.e. cells T8, R7, R7-1, R7-2, R7S, R7S-1, R7S-2,R7S-3, R7S-1s, R7S-2s, and R7S-3s.

Concretely, in accordance with the pulse-chase labeling andimmunoprecipitation method with ³⁵S-methionine, expression levels inextracellular matrix fibronectin (FN), integrin beta 1 (integrin β1)transmembrane receptor, and endoplasmic reticulum resident chaperone Bipfor each of the cells. The results on the cells T8, pLPCX, asR70, asR56,asR46, and sR70 are shown in FIG. 4-a.

It could be seen from FIG. 4-a that expression levels of Bip are loweredwith passage of time in the cells asR70, the cells asR56, the cellsasR46, and the cells pLPCX (see the graph circumscribed with box in FIG.4-a). On the other hand, expression levels of the FN and the integrin β1were shown to have the same tendencies as signal-independent actin orglucose 6-phosphate dehydrogenase, so that hardly any changes were foundtherewith. While expression levels of FN and integrin β1 were not foundto show any changes because of high productivity in the cells,expression levels of Bip were found to show some changes; therefore, itis suggested that Bip synthesis is inhibited in ribosomes of the cellsasR70, the cells asR56, and the cells asR46.

In addition, in order to further study the influences of Bip in moredetail, the same studies were conducted on the cytoplasmic proteins.Concretely, 30 μg of a cytoplasmic protein was separated by 10%SDS/PAGE, and Bip, eIF2a (eukaryotic initiation factor 2a),phosphorylated-eIF2a (P-eIF2a), and ATF4 (activating transcriptionfactor 4) were immunoblotted. The results on the cells T8, pLPCX, asR70,asR56, asR46, and sR70 are shown in FIG. 4-b, and the results on thecells T8, R7, R7-1, R7-2, R7S, R7S-1, R7S-2, R7S-3, R7S-1s, R7S-2s,R7S-3s are shown in FIG. 4-c. Here, if the Bip synthesis is inhibited, aso-called endoplasmic reticulum stress response is induced, in whicheIF2a is phosphorylated to inhibit protein synthesis, and thephosphorylated eIF2a initiates the translation of ATF4.

According to FIGS. 4-b and -c, the expression levels of Bip and eIF2awere nearly of the same level in all the cells, but expression ofphosphorylated eIF2a was more markedly confirmed in the cells asR46, thecells R7, the cells R7-1, and the cells R7-2, and induction of ATF4accompanying therewith was also confirmed. It is suggested from theabove that asR46 and asR70 inhibit Bip synthesis, thereby inducing theendoplasmic reticulum stress response.

Example 7 Screening of Anticancer Agent

Cells introduced with hTERT, SV40ST, and a human 28S rRNA-derivedoligonucleotide are cultured in a cell culture equipment, and a solutionprepared by dissolving a candidate compound in a solvent is addedthereto, and the cells are further cultured. On the other hand, using agroup with no addition of a candidate compound as a control, in caseswhere changes such as transformed morphological losses, induction ofapoptosis, and losses of anchorage-independent growth of the cells arefound to be greater than the group with no addition, the candidatecompound added to the cells can be judged to have a high possibility ofpossessing an action as an anticancer agent.

The method for preparing neoplastically transformed cells fromhuman-derived cells of the present invention can be utilized when avariety of human normal cells are induced to be neoplasticallytransformed in order to elucidate cancer onset mechanisms, so that themethod can be effectively utilized in search of target molecules for anew medicament.

The present invention being thus described, it will be obvious that thesame may be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1 of the Sequence Listing is an antisense oligonucleotideasR70 derived from human 28S rRNA;

SEQ ID NO: 2 of the Sequence Listing is an antisense oligonucleotideasR46 derived from human 28S rRNA;

SEQ ID NO: 3 of the Sequence Listing is a partial nucleotide sequence oftransforming RNA;

SEQ ID NO: 4 of the Sequence Listing is a partial nucleotide sequence ofhuman 28S rRNA;

SEQ ID NO: 5 of the Sequence Listing is a partial nucleotide sequence ofhuman SRP RNA;

SEQ ID NO: 6 of the Sequence Listing is an antisense oligonucleotideasR56 derived from human 28S rRNA;

SEQ ID NO: 7 of the Sequence Listing is an oligonucleotide sR70 derivedfrom human 28S rRNA;

SEQ ID NO: 8 of the Sequence Listing is a partial nucleotide sequence ofhuman 28S rRNA;

SEQ ID NO: 9 of the Sequence Listing is a modified sequence of thepartial nucleotide sequence of transforming RNA;

SEQ ID NO: 10 of the Sequence Listing is a primer for pLPCX-retroF;

SEQ ID NO: 11 of the Sequence Listing is a primer for asR70 cDNA;

SEQ ID NO: 12 of the Sequence Listing is a primer for pLPCX-seqR;

SEQ ID NO: 13 of the Sequence Listing is a primer for β-actin; and

SEQ ID NO: 14 of the Sequence Listing is a primer for β-actin.

1. A method for preparing a neoplastically transformed cell from ahuman-derived cell, the method comprising introducing a human telomerasecatalytic subunit (hTERT) gene, an SV40 small T antigen (SV40ST) gene,and an antisense oligonucleotide derived from a human 28S rRNA into thehuman-derived cell.
 2. The method according to claim 1, wherein theantisense oligonucleotide derived from a human 28S rRNA is a firstoligonucleotide comprising a nucleotide sequence of SEQ ID NO: 1, or asecond oligonucleotide comprising a sequence having 70% or more homologyto the nucleotide sequence of SEQ ID NO: 1, wherein a neoplastictransformation efficiency of the second oligonucleotide is equivalent toa neoplastic transformation efficiency of the first oligonucleotide. 3.The method according to claim 1, wherein the antisense oligonucleotidederived from a human 28S rRNA is a first oligonucleotide comprising anucleotide sequence of SEQ ID NO: 2, or a second oligonucleotidecomprising a sequence having 70% or more homology to the nucleotidesequence of SEQ ID NO: 2, wherein a neoplastic transformation efficiencyof the second oligonucleotide is equivalent to a neoplastictransformation efficiency of the first oligonucleotide.
 4. The methodaccording to claim 1, wherein the human-derived cell is a humanfibroblast cell.
 5. A method for introducing a gene that neoplasticallytransforms a human-derived cell, the method comprising incorporating ahuman telomerase catalytic subunit (hTERT) gene, an SV40 small T antigen(SV40ST) gene, and an antisense oligonucleotide derived from a human 28SrRNA into one or more vectors, and introducing the vector into thehuman-derived cell.
 6. A neoplastically transformed cell obtained by themethod of claim
 1. 7. A kit that performs the method of claim 1,comprising the human telomerase catalytic subunit (hTERT) gene, the SV40small T antigen (SV40ST) gene, and the antisense oligonucleotide derivedfrom a human 28S rRNA.
 8. A method for screening an anticancer agent,comprising culturing a first neoplastically transformed cell obtained bythe method of claim 1 in the presence of a candidate compound, culturinga second neoplastically transformed cell obtained by the method of claim1 in the absence of the candidate compound, measuring a degree ofinhibition of the first neoplastically transformed cell and a degree ofinhibition of the second neoplastically transformed cell, and judgingthat the candidate compound has a high possibility of having an actionas an anticancer agent when the degree of inhibition of the firstneoplastically transformed cell is greater than the degree of inhibitionof the second neoplastically transformed cell.
 9. A method for screeningan anticancer agent, comprising culturing a first human-derived cellinto which the genes are introduced according to the method of claim 5in the presence of a candidate compound, culturing a secondhuman-derived cell into which the genes are introduced according to themethod of claim 5 in the absence of a candidate compound, measuring adegree of inhibition of neoplastic transformation of the firsthuman-derived cell and a degree of inhibition of neoplastictransformation of the second human-derived cell, and judging that thecandidate compound has a high possibility of having an action as ananticancer agent when the degree of inhibition of the firsthuman-derived cell is greater than the degree of inhibition of thesecond human-derived cell.
 10. The method according to claim 1, whereinthe antisense oligonucleotide derived from a human 28S rRNA comprises anucleotide sequence of SEQ ID NO:
 1. 11. The method according to claim1, wherein the antisense oligonucleotide derived from a human 28S rRNAcomprises a nucleotide sequence of SEQ ID NO:
 2. 12. The methodaccording to claim 1, wherein the antisense oligonucleotide derived froma human 28S rRNA comprises a nucleotide sequence of SEQ ID NO:
 6. 13.The method according to claim 1, wherein the antisense oligonucleotidederived from a human 28S rRNA consists of a nucleotide sequence of SEQID NO:
 1. 14. The method according to claim 1, wherein the antisenseoligonucleotide derived from a human 28S rRNA consists of a nucleotidesequence of SEQ ID NO:
 2. 15. The method according to claim 1, whereinthe antisense oligonucleotide derived from a human 28S rRNA consists ofa nucleotide sequence of SEQ ID NO:
 6. 16. The method according to claim1, wherein the antisense oligonucleotide derived from a human 28S rRNAcomprises a sequence having 70% or more homology to a nucleotidesequence of SEQ ID NO:
 1. 17. The method according to claim 1, whereinthe antisense oligonucleotide derived from a human 28S rRNA comprises asequence having 70% or more homology to a nucleotide sequence of SEQ IDNO:
 2. 18. The method according to claim 1, wherein the antisenseoligonucleotide derived from a human 28S rRNA has a length of 40 to 200.19. The method according to claim 1, wherein the antisenseoligonucleotide derived from a human 28S rRNA has a length of 46 to 70.20. The method according to claim 1, wherein the introducing is by anelectroporation method, a calcium phosphate method, or a lipofectionmethod.